Rajeev Chaudhary

Rapid multicomponent T2 analysis of the articular cartilage of the human knee joint at 3.0T

To determine the feasibility of using multicomponent‐driven equilibrium single‐shot observation of T1 and T2 (mcDESPOT) for evaluating the human knee joint at 3.0T and to investigate depth‐dependent and regional‐dependent variations in multicomponent T2 parameters within articular cartilage.

Articular cartilage zonal differentiation via 3D Second-Harmonic Generation imaging microscopy

Abstract Purpose: The collagen structure throughout the patella has not been thoroughly investigated by 3D imaging, where the majority of the existing data come from histological cross sections. It is important to have a better understanding of the architecture in normal tissues, where this could then be applied to imaging of diseased states. Methods: To address this shortcoming, we investigated the combined use of collagen-specific Second-Harmonic Generation (SHG) imaging and measurement of bulk optical properties to characterize collagen fiber orientations of the histologically defined zones of bovine articular cartilage. Forward and backward SHG intensities of sections from superficial, middle and deep zones were collected as a function of depth and analyzed by Monte Carlo simulations to extract the SHG creation direction, which is related to the fibrillar assembly. Results: Our results revealed differences in SHG forward–backward response between the three zones, where these are consistent with a previously developed model of SHG emission. Some of the findings are consistent with that from other modalities; however, SHG analysis showed the middle zone had the most organized fibril assembly. While not distinct, we also report bulk optical property values for these different zones within the patella. Conclusions: Collectively, these results provide quantitative measurements of structural changes at both the fiber and fibril assembly of the different cartilage zones and reveals structural information not possible by other microscope modalities. This can provide quantitative insight to the collagen fiber network in normal cartilage, which may ultimately be developed as a biomarker for osteoarthritis.

Accuracy of model-based tracking of knee kinematics and cartilage contact measured by dynamic volumetric MRI

The purpose of this study was to determine the accuracy of knee kinematics and cartilage contact measured by volumetric dynamic MRI. A motor-actuated phantom drove femoral and tibial bone segments through cyclic 3D motion patterns. Volumetric images were continuously acquired using a 3D radially undersampled cine spoiled gradient echo sequence (SPGR-VIPR). Image data was binned based on position measured via a MRI-compatible rotary encoder. High-resolution static images were segmented to create bone models. Model-based tracking was performed by optimally registering the bone models to the volumetric images at each frame of the SPGR-VIPR series. 3D tibiofemoral translations and orientations were reconstructed, and compared to kinematics obtained by tracking fiducial markers. Imaging was repeated on a healthy subject who performed cyclic knee flexion-extension. Cartilage contact for the subject was assessed by measuring the overlap between articular cartilage surfaces. Model-based tracking was able to track tibiofemoral angles and translations with precisions less than 0.8° and 0.5mm. These precisions resulted in an uncertainty of less than 0.5mm in cartilage contact location. Dynamic SPGR-VIPR imaging can accurately assess in vivo knee kinematics and cartilage contact during voluntary knee motion performed in a MRI scanner. This technology could facilitate the quantitative investigation of links between joint mechanics and the development of osteoarthritis.

Multicomponent T2 analysis of articular cartilage with synovial fluid partial volume correction

To investigate the use of a three‐pool model to account for the confounding effects of synovial fluid on multicomponent T2 analysis of articular cartilage using Multicomponent Driven Equilibrium Single Shot Observation of T1 and T2 (mcDESPOT).

Quantitative Magnetic Resonance Imaging of the Articular Cartilage of the Knee Joint

Osteoarthritis is characterized by a decrease in the proteoglycan content and disruption of the highly organized collagen fiber network of articular cartilage. Various quantitative magnetic resonance imaging techniques have been developed for noninvasive assessment of the proteoglycan and collagen components of cartilage. These techniques have been extensively used in clinical practice to detect early cartilage degeneration and in osteoarthritis research studies to monitor disease-related and treatment-related changes in cartilage over time. This article reviews the role of quantitative magnetic resonance imaging in evaluating the composition and ultrastructure of the articular cartilage of the knee joint.

Periosteal Fiber Transection During Periosteal Procedures Is Crucial to Accelerate Growth in the Rabbit Model

BackgroundDisruption of the periosteum has been used to explain overgrowth after long bone fractures. Clinically, various periosteal procedures have been reported to accelerate growth with varied results. Differences between procedures and study populations, in these prior studies, make drawing conclusions regarding their effectiveness difficult.Questions/purposesThe purpose of this study was to (1) determine if all reported periosteal procedures accelerate growth and increase the length of bones; (2) study the relative duration of these growth-accelerating effects at two time points; and (3) identify the periosteal procedure that results in the most growth.MethodsPeriosteal stripping (N = 8), periosteal transection (N = 8), periosteal resection (N = 8), (and) full periosteal release (N = 8) were performed on the tibiae of skeletally immature rabbits. Tibiae were collected 2 weeks postoperatively. The tibiae of additional cohorts of periosteal transection (N = 8), periosteal resection (N = 8), full periosteal release (N = 8), and repetitive periosteal transection (N = 8) were collected 8 weeks postoperatively. The contralateral tibiae served as an operative sham control in all cohorts. Fluorochrome bone labeling was used to measure growth rates, whereas high-resolution Faxitron imaging was performed to measure tibial lengths. Comparisons were then made between (1) experimental and sham controls; and (2) different procedures. Eight additional nonsurgical animals were included as age-matched controls.ResultsGrowth (in microns) was accelerated at the proximal tibial physis on the tibia undergoing the periosteal surgical procedures versus the contralateral control limb after the transection (411 ± 27 versus 347 ± 18, p < 0.001 [mean ± SD]), resection (401 ± 33 versus 337 ± 31, p < 0.001), and full periosteal release (362 ± 45 versus 307 ± 33, p < 0.001), 2 weeks after the index procedure. Conversely, the periosteal stripping cohort trended toward less growth (344 ± 35) than the controls (356 ± 25; p = 0.08). No differences were found between limbs in the nonoperative controls. Tibial lengths for the experimental tibiae were longer at 2 weeks in the transection (1.6 ± 0.4 mm, p < 0.001), resection (1.6 ± 0.9 mm, p = 0.03), and full periosteal release (1.7 ± 0.5 mm, p < 0.001), whereas negligible differences were found between the tibiae of the nonoperative controls (0.13 ± 0.7 mm, p = 0.8) and stripping cohorts (0.10 ± 0.6 mm, p = 0.7). At 8 weeks, growth acceleration ceased at the proximal tibial physes in the transection cohort (174 ± 11 versus 176 ± 21, p = 0.8), and the control limbs actually grew faster than the experimental limbs after resection (194 ± 24 versus 178 ± 23, p = 0.02) and full periosteal release (193 ± 16 versus 175 ± 19, p < 0.01) cohorts. Growth rates were increased over control limbs, only in the repetitive transection cohort (190 ± 30 versus 169 ± 19, p = 0.01) at 8 weeks. Tibial lengths for the experimental tibiae remained longer at 8 weeks in the transection (1.4 ± 0.70 mm, p < 0.001), resection (2.2 ± 0.82 mm, p < 0.001), full periosteal release (1.6 ± 0.42 mm, p < 0.001), and repetitive periosteal transection (3.3 ± 1.1 mm, p < 0.001), whereas negligible differences were found between the tibiae of the nonoperative controls (−0.08 ± 0.58 mm, p = 0.8). Comparing the procedures at 2 weeks postoperatively, no differences were found in tibial lengths among the transection (2.1% ± 0.5% increase), resection (2.1% ± 1.1% increase), and full periosteal release (2.1% ± 0.6 %); however, all three demonstrated greater increased growth when compared with the stripping cohort (−0.10% ± 0.7%; p < 0.05). At 8 weeks no differences could be found between increased tibial lengths among the transection (1.5% ± 0.7%), resection (2.3% ± 0.9%), and full periosteal release (1.7% ± 0.4%). The repetitive transection produced the greatest over length increase (3.5% ± 1%), and this was greater than the acceleration generated by the single resection (p < 0.001) or the full periosteal release (p = 0.001). All four demonstrated an increase greater than the nonoperative control (0.09% ± 0.6%; p < 0.05).ConclusionsTransection of the longitudinally oriented periosteal fibers appears critical to accelerate growth in a rabbit model.Clinical RelevanceThese findings in an animal model support previous claims that limb overgrowth occurs as the result of periosteal disruption. Based on these findings in rabbits, we believe that less invasive procedures like periosteal transection are a promising avenue to explore in humans; clinical studies should seek to determine whether it is equally effective as more invasive procedures and its role as an adjunct to guided growth or distraction osteogenesis.

Advanced quantitative imaging and biomechanical analyses of periosteal fibers in accelerated bone growth.

PURPOSE The accepted mechanism explaining the accelerated growth following periosteal resection is that the periosteum serves as a mechanical restraint to restrict physeal growth. To test the veracity of this mechanism we first utilized Second Harmonic Generation (SHG) imaging to measure differences of periosteal fiber alignment at various strains. Additionally, we measured changes in periosteal growth factor transcription. Next we utilized SHG imaging to assess the alignment of the periosteal fibers on the bone both before and after periosteal resection. Based on the currently accepted mechanism, we hypothesized that the periosteal fibers adjacent to the physis should be more aligned (under tension) during growth and become less aligned (more relaxed) following metaphyseal periosteal resection. In addition, we measured the changes in periosteal micro- and macro-scale mechanics. METHODS 30 seven-week old New Zealand White rabbits were sacrificed. The periosteum was imaged on the bone at five regions using SHG imaging. One centimeter periosteal resections were then performed at the proximal tibial metaphyses. The resected periosteal strips were stretched to different strains in a materials testing system (MTS), fixed, and imaged using SHG microscopy. Collagen fiber alignment at each strain was then determined computationally using CurveAlign. In addition, periosteal strips underwent biomechanical testing in both circumferential and axial directions to determine modulus, failure stress, and failure strain. Relative mRNA expression of growth factors: TGFβ-1, -2, -3, Ihh, PTHrP, Gli, and Patched were measured following loading of the periosteal strips at physiological strains in a bioreactor. The periosteum adjacent to the physis of six tibiae was imaged on the bone, before and after, metaphyseal periosteal resection, and fiber alignment was computed. One-way ANOVA statistics were performed on all data. RESULTS Imaging of the periosteum at different regions of the bone demonstrated complex regional differences in fiber orientation. Increasing periosteal strain on the resected strips increased periosteal fiber alignment (p<0.0001). The only exception to this pattern was the 10% strain on the tibial periosteum, which may indicate fiber rupture at this non-physiologic strain. Periosteal fiber alignment adjacent to the resection became less aligned while those adjacent to the physes remained relatively unchanged before and after periosteal resection. Increasing periosteal strain on the resected strips increased periosteal fiber alignment (p<0.0001). The only exception to this pattern was the 10% strain on the tibial periosteum, which may indicate fiber rupture (and consequent retraction) at this non-physiologic strain. Increasing periosteal strain revealed a significant increase in relative mRNA expression for Ihh, PTHrP, Gli, and Patched, respectively. CONCLUSION Periosteal fibers adjacent to the growth plate do not appear under tension in the growing limb, and the alignments of these fibers remain unchanged following periosteal resection. SIGNIFICANCE The results of this study call into question the long-accepted role of the periosteum acting as a simple mechanical tether restricting growth at the physis.

Polarization-resolved second harmonic generation imaging of human ovarian cancer

Abstract. Remodeling of the extracellular matrix in human ovarian cancer can be manifested in increased collagen concentration, changes in alignment within fibrils/fibers and/or up-regulation of different collagen isoforms. We used pixel-based second harmonic generation (SHG) polarization microscopy analyses to probe these molecular changes in human ovarian tissues [normal stroma, benign tumors, and high-grade serous (HGS) tumors] by: (i) determination of the α-helical pitch angle via the single-axis molecular model, (ii) collagen alignment within fibrils via SHG anisotropy, and (iii) chirality via SHG circular dichroism (SHG-CD). Pixel approaches are required due to the complex structure of the matrix that lacks a high degree of fiber alignment. The largest differences in the helical pitch angle were between normal stroma and benign tumors, consistent with gene expression showing the Col III isoform is up-regulated in the latter. The data were not consistent with up-regulation of Col III in HGS tumors as previous reports have suggested. The different tissues also displayed differing SHG anisotropies and SHG-CD responses, consistent with either Col III incorporation or randomization of Col I alignment within benign and malignant tumors. Additionally, the high-grade tumors displayed higher collagen concentration, where this desmoplasia is consistent with the higher fiber density in these tissues. These results collectively indicate that the fibril assemblies are distinct in all tissues, where these differences likely result from the synthesis of collagen rather than remodeling of existing collagen. Importantly, these analyses are label-free and interrogate subresolution collagen structure on intact tissues, without the need for conventional structural biology tools.

Pixel based SHG probes of extracellular matrix (ECM) alterations in ovarian cancer (Conference Presentation)

Remodeling of the extracellular matrix in human ovarian cancer, can be reflected in increased collagen concentration, changes in alignment and/or up-regulation of different collagen isoforms, including Col III. Using fibrillar gel models, we demonstrate that Col I and Col III can be quantitatively distinguished by 3 distinct SHG polarization specific metrics: i) determination of helical pitch angle via the single axis molecular model, ii) dipole alignment via anisotropy, and iii) chirality via SHG circular dichroism (SHG-CD). These sub-resolution differentiations are possible due to differences in the α helix angles of the two isoforms, which co-mingle in the same fibrils. We also investigated the mechanism of the SHG-CD response and show that unlike conventional CD, it is dominated by electric dipole interactions and is consistent with the two state SHG model. We further applied these 3 polarization resolved analyses to human normal, high risk, benign tumors, and malignant human ovarian tissues. We found that these tissues could all be differentiated by these metrics, where high grade tissues had analogous α-helical pitch angles to the in the Col I/Col III gel model. This confirms literature suggestions based on immunofluorescence and gene expression that Col III is up-regulated in high grade ovarian cancers. The different tissues also displayed differing anisotropies, indicating the fibril assemblies are distinct and likely do not result from remodeling of existing collagen but synthesis of new collagen. Importantly, these SHG polarization methods provide structural information not otherwise possible and can serve as label-free biomarkers for ovarian and other cancers.

Advanced quantitative imaging of musculoskeletal disorders (Conference Presentation)

Previous studies have shown that bone growth acceleration can occur in many animal species after periosteal resection (removal of a strip of periosteum) with minimum morbidity. This has numerous clinical applications, including treatment of limb length differences. Here we use Second Harmonic Generation (SHG) imaging microscopy to evaluate changes in collagen architecture reflective of the different strains the periosteum may encounter during bone growth. Specifically, we image rabbit tibial periosteum strips at -20%, 0%, 5%, and 10% strains. We first quantify these changes using the SHG creation ratio (Forward/Backward) or the initially emitted SHG directionality to provide information on the fibril level of assembly. The in situ (i.e. physiological) strain had the highest creation ratio compared to the non-in situ strains of -20%, 5%, and 10%, which were shown to be significantly different via RCBD statistical analysis. These trends are consistent with SHG phasematching considerations, where more organized fibrils/fibers result in primarily forward emitted components, which here is the physiological strain. We further use the relative SHG conversion efficiency to assess the tissue structure under strain, where this results from the combination of collagen concentration and organization. The 0% strain SHG conversion efficiency was significantly higher than all other strains, where this is expected as the fibers have the highest local density and organization, and is consistent with the emission directionality results. Importantly, due to the underlying physical process, the label-free SHG imaging modality can non-invasively monitor the effect of treatments for bone growth and other orthopedic disorders.